The process of bisulfite treatment denatures 50 ng of genomic DNA into single-stranded DNA (ssDNA). The protocol converts bisulfite-treated, ssDNA into an Illumina sequencing library. Post-bisulfite conversion method ensures that all ssDNA fragments are captured during the procedure, eliminating sample loss.
Bisulfite Sequencing
Technologies
SOLVING THE GENOME'S SECOND CODE
DNA methylation is a fundamental mechanism of the epigenetic regulation of gene activity. It has been shown that DNA methylation plays an important role in a wide variety of biological processes, including silencing of transposable elements, stem cell differentiation, embryonic development, genomic imprinting and inflammation. In addition, alteration of methylation patterns has been identified in many diseases, inflammation and neurological disorders.
By combining bisulfite treatment of genomic DNA with Next-Gen sequencing it is possible to sensitively measure cytosine methylation on a genome-wide scale within specific sequence contexts.
However, the analysis of each methylated cytosin in the genome might be quite expensive. By using restriction enzymes and bisulfite sequencing it is possible to enrich for the areas of the genome that have higher CpG content. This approach, termed RRBS-seq (Reduced Representation Bisulfite Sequencing), reduces the amount of nucleotides needed to be sequenced to 1% of genome size, allowing for a cost-effective single-base-pair resolution of methylated cytosines.
The third alternative is targeted bisulfite sequencing, which is able to specifically capture selected genomic regions of interest associated with a disease or phenotype.
IGATech is adopting conversion protocol to analyze both 5mC and 5hmC in the same experiment.
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WHOLE GENOME BS-seq (WGBS-seq)
REDUCED REPRESENTATION BS-seq (RRBS-seq)
RRBS is used to generate single base resolution DNA methylation (5-methylC) information across a genomic sample, starting form as little as 100 ng of high-quality genomic DNA.
The current approach utilizes the methylation insensitive restriction enzymes MspI for mammals and TaqI for plant species.
The introduction of integrated molecular tag during the sample processing enables the removal of non-unique reads from the dataset.
TARGETED BISULFITE SEQUENCING (human only)
Bisulfite conversion sequencing can be done with targeted methods such as target enrichment.
The protocols used are fully customized and enable the targeting of selected genomic regions from bisulfite treated genomic DNA in a single workflow, in order to identify specific regions in the genome for methylation variation assessment.
As final analysis, it is possible to target selected human genomic regions from bisulfite treated genomic DNA in a single workflow.
Our team is always available to consult with you on study design to ensure correct sequencing and bioinformatics strategies are used to meet your goals.
BIOINFORMATICS SERVICES
We offer state-of-art analysis pipelines for WGBS-seq, RRBS-seq and Targeted BS-seq. The workflow includes:
- Quality control
- Post-sequencing estimation of conversion rate using Lambda spike-in
- Alignment and deduplication
- Methylation calling at single cytosine level
- Whole genome methylation statistics (including distribution of methylation in the CG, CHG and CHH context in plants)
- Visualization of methylation distribution across the genome using CIRCOS image
- Identification of Differentially Methylated Regions (DMRs) between samples
- Annotation of DMRs